Project Summary/Abstract As the site of both negative selection of developing thymocytes and generation of regulatory T cells (Tregs), the thymus plays a critical role in the multi-layered network of immune tolerance. The fate of a developing T cell is dependent on the affinity of the interaction between the TCR and the peptide-MHC complex it recognizes, with the highest affinity interactions resulting in deletion or Treg induction and lower affinity interactions resulting in T cell survival and differentiation. The Autoimmune Regulator (Aire) is a major transcriptional regulator of peripheral self-antigen expression within the thymus, and loss of Aire leads to defects in negative selection due to reduced or absent thymic antigen expression in specialized medullary thymic epithelial cells (mTECs). Several lines of evidence point to this process as playing an important role in the pathogenesis of type 1 diabetes. Here, we will use type 1 diabetes as a model disease system to further unravel how insulin-specific T cells arise from the thymus that are both T effectors and T regulatory cells. Our specific aims are: AIM1: Define the role of Aire and thymic insulin expression on the thymic deletion of insB(9-23) specific T cells. AIM2: Define the repertoire and specificity of thymic Foxp3+ T regulatory cells selected by Aire-expressing cells. These studies will be performed in close collaboration with PPG projects 2 (Bluestone) and 3 (Kappler) and will help improve our understanding of how the T cell repertoire is shaped in the thymus in the setting of type 1 diabetes.